Ovine CD16+/CD14− blood lymphocytes present all the major characteristics of natural killer cells

Natural killer (NK) cells have a key role in the innate immune response against pathogens because of their cytotoxic properties and production of interferon-gamma (IFN-γ). Some insight into ruminant NK cell biology has been gained through the characterization of bovine NK cells as NKp46+/CD3− cells. However, ovine NK cells have been little studied because of the lack of specific antibodies. Most NK cells in humans and cattle express CD16. We found that an antibody against human CD16 that cross-reacts with bovine NK cells also recognizes cell populations in ovine peripheral blood mononuclear cells. Using double labelling with CD14 revealed the same profile as described in other species, and we identified a putative NK cell population. We therefore sorted this ovine CD16+/CD14− cell population and tested it for NK cell characteristics. More than 80% of sorted CD16+/CD14− cells expressed perforin. After a week of culture in the presence of IL-2 and IL-15, ovine CD16+/CD14− cells had become large cells with intra-cytoplasmic granules containing perforin, and the vast majority displayed an activated CD2−/low/CD25+/CD8+ phenotype, as observed for bovine NKp46+/CD3− cells. Moreover, these cells expressed transcripts for the NKp46 receptor, and were cytotoxic in a CD16-mediated redirected lysis assay against a murine cell line, P815, and in a direct lysis assay against the ovine cell line, IDO5. Finally, ovine CD16+/CD14− cells having expanded for 7 days in culture secreted IFN-γ in response to IL-12 in a dose-dependent manner. Taken together, these findings led us to conclude that the ovine CD16+/CD14− lymphocyte sub-population displays the phenotype and functional characteristics of NK cells

Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo

<p>Abstract</p> <p>Background</p> <p>Telomerase activation, a critical step in cell immortalization and oncogenesis, is partly regulated by alternative splicing. In this study, we aimed to use the Marek's disease virus (MDV) T-cell lymphoma model to evaluate TERT regulation by splicing during lymphomagenesis <it>in vivo</it>, from the start point to tumor establishment.</p> <p>Results</p> <p>We first screened cDNA libraries from the chicken MDV lymphoma-derived MSB-1 T- cell line, which we compared with B (DT40) and hepatocyte (LMH) cell lines. The chTERT splicing pattern was cell line-specific, despite similar high levels of telomerase activity. We identified 27 alternative transcripts of chicken TERT (chTERT). Five were in-frame alternative transcripts without <it>in vitro </it>telomerase activity in the presence of viral or chicken telomerase RNA (vTR or chTR), unlike the full-length transcript. Nineteen of the 22 transcripts with a premature termination codon (PTC) harbored a PTC more than 50 nucleotides upstream from the 3' splice junction, and were therefore predicted targets for nonsense-mediated decay (NMD). The major PTC-containing alternatively spliced form identified in MSB1 (ie10) was targeted to the NMD pathway, as demonstrated by UPF1 silencing. We then studied three splicing events separately, and the balance between in-frame alternative splice variants (d5f and d10f) plus the NMD target i10ec and constitutively spliced chTERT transcripts during lymphomagenesis induced by MDV indicated that basal telomerase activity in normal T cells was associated with a high proportion of in-frame non functional isoforms and a low proportion of constitutively spliced chTERT. Telomerase upregulation depended on an increase in active constitutively spliced chTERT levels and coincided with a switch in alternative splicing from an in-frame variant to NMD-targeted variants.</p> <p>Conclusions</p> <p>TERT regulation by splicing plays a key role in telomerase upregulation during lymphomagenesis, through the sophisticated control of constitutive and alternative splicing. Using the MDV T-cell lymphoma model, we identified a chTERT splice variant as a new NMD target.</p

Fluorescent tagging of VP22 in N-terminus reveals that VP22 favors Marek’s disease virus (MDV) virulence in chickens and allows morphogenesis study in MD tumor cells

Marek’s disease virus (MDV) is an alpha-herpesvirus causing Marek’s disease in chickens, mostly associated with T-cell lymphoma. VP22 is a tegument protein abundantly expressed in cells during the lytic cycle, which is essential for MDV spread in culture. Our aim was to generate a pathogenic MDV expressing a green fluorescent protein (EGFP) fused to the N-terminus of VP22 to better decipher the role of VP22 in vivo and monitor MDV morphogenesis in tumors cells. In culture, rRB-1B EGFP22 led to 1.6-fold smaller plaques than the parental virus. In chickens, the rRB-1B EGFP22 virus was impaired in its ability to induce lymphoma and to spread in contact birds. The MDV genome copy number in blood and feathers during the time course of infection indicated that rRB-1B EGFP22 reached its two major target cells, but had a growth defect in these two tissues. Therefore, the integrity of VP22 is critical for an efficient replication in vivo, for tumor formation and horizontal transmission. An examination of EGFP fluorescence in rRB-1B EGFP22-induced tumors showed that about 0.1% of the cells were in lytic phase. EGFP-positive tumor cells were selected by cytometry and analyzed for MDV morphogenesis by transmission electron microscopy. Only few particles were present per cell, and all types of virions (except mature enveloped virions) were detected unequivocally inside tumor lymphoid cells. These results indicate that MDV morphogenesis in tumor cells is more similar to the morphorgenesis in fibroblastic cells in culture, albeit poorly efficient, than in feather follicle epithelial cell

Modulation de la résistance aux antiparasitaires par les stérols chez un nématode <em>Haemonchus contortus</em>, parasite des petits ruminants

National audienc

Mise en place d'un test de criblage rapide pour évaluer l’effet antiparasitaire de molécules vis-à-vis du développement d'<em>Eimeria tenella</em> in vitro

National audienc

Nouvelle stratégie de modulation par les stérols de la résistance aux antihelminthiques médiée par les P-glycoprotéines chez Haemonchus contortus

National audienc

ATP content and flow cytometric evaluation of mitochondrial activity and membrane integrity in fresh and cryopreserved trout spermatozoa: Effect of different cryoprotectants

32. Annual meeting of the Society for Cryobiology. Résumé 116International audienc

Impact of IFNyR and IL-17RA on neutrophils recruitment during mycobacterial infections

National audienc

ATP content and flow cytometric evaluation of mitochondrial activity and membrane integrity in fresh and cryopreserved trout spermatozoa: Effect of different cryoprotectants

32. Annual meeting of the Society for Cryobiology. Résumé 116International audienc